Easy,Rapid,and Cost-Effective Methods for Identifying Carriers of Recurrent GJB2 Mutations Causing Nonsyndromic Hearing Impairment in the Greek Population

Abstract

A variety of techniques have been developed for screening the GJB2 gene for known and unknown mutations, especially the most common mutation in the Caucasian population, the c.35delG. Other mutations that have been so far characterized in the GJB2 gene seem to have different geographical distributions, and therefore there is an interest in identifying recurrent mutations specific for each population and developing easy and rapid screening techniques. Here we present easy screening protocols for already identified recurrent mutations in the Greek population. Developing easy, rapid, and cost-effective screening methods will facilitate the detection of GJB2 recurrent mutation carriers, at large, in the Greek population.

Introduction

Approximately 1 in 1000 children suffers from severe or profound hearing loss at birth or during early childhood (prelingual deafness) (Morton, 1991). At least 50% of deafness cases have been related to genetic causes. Among hereditary nonsyndromic deafness, autosomal recessive inheritance predominates, accounting for about 80% of the cases (Zelante et al., 1997), and up to date about 50 genes have been identified to be involved. Mutations in the GJB2 gene represent a major cause of prelingual, nonsyndromic, recessive deafness, as they are responsible for up to 40% of such cases in many populations (Denoyelle et al., 1997; Kelley et al., 1998; Scott et al., 1998; Rabionet et al., 2000a; Kelsell et al., 2001; Kenneson et al., 2002). The autosomal recessive forms of deafness are usually the most severe and are almost exclusively due to cochlear defects (sensorineural deafness), in contrast to the syndromic forms of deafness, which in most cases are conductive (external and/or middle ear developmental defects) or mixed (Petit, 1996). Around 90 GJB2 mutations have so far been reported to be associated with recessive, nonsyndromic hearing loss (The Connexin-deafness homepage, http://davinci.crg.es/deafness/). One specific mutation, c.35delG, consists of a deletion of guanine in a sequence of six guanines leading to a frameshift and premature stop of the synthesis of Cx26, the GJB2 gap junction protein (Denoyelle et al., 1997). c.35delG homozygosity is severe or profound in most cases, but it can also be mild or moderate (Snoeckx et al., 2005). The hearing loss is usually prelingual and nonprogressive, but some cases of progression have been reported (Cohn et al., 1999; Denoyelle et al., 1999). The relative contribution of the GJB2 gene to nonsyndromic, prelingual deafness varies from 0% to 40% in the populations studied (Petersen and Willems, 2006), demonstrating genetic heterogeneity. Previous publications report a high prevalence of the c.35delG mutation in Caucasians (Petit, 1996; Denoyelle et al., 1997; Estivill et al., 1998; Kelley et al., 1998), with Greece showing a higher proportion with 1/3 of deafness cases resulting from the c.35delG mutation (Pampanos et al., 2002). High carrier frequency (3.5%) of the mutation in the Greek population has been previously described (Antoniadi et al., 1999).

So far, in our laboratory, all patients with sensorineural, nonsyndromic hearing loss have been tested for the common GJB2 c.35delG mutation by amplification refractory mutation system (ARMS)-PCR, and direct sequencing of the entire coding region of the gene was performed in all heterozygotes for a possible detection of a second mutation. However, in terms of carrier screening of a healthy population, at large, direct sequencing can be very highly priced and time consuming. For this reason, we developed easy, rapid, and cost-effective screening techniques for the detection of recurrent GJB2 mutations in the Greek population. Tests were performed on a selected group of 28 familial cases with nonsyndromic sensorineural hearing impairment and on 200 controls with normal hearing.

Materials and Methods

Materials

A total of 200 unrelated Greek subjects without any noticeable hearing loss were recruited. The controls consisted of healthy parents of children referred to our department for analysis of syndromic mental retardation. Further, 51 unrelated patients with nonsyndromic prelingual sensorineural deafness, who had a positive family history of prelingual deafness, were referred from major centers for childhood deafness in Greece. The hearing impairment was bilateral in all cases, and severity ranged from mild to profound. We included 28 probands in our study from these cases. All GJB2 c.35delG homozygotes (n = 18) or compound heterozygotes (n = 2) with other disease-causing mutations previously identified by ARMS-PCR and direct genomic sequencing were excluded from the present study. The 28 probands consisted of one c.35delG heterozygote and 27 patients without the c.35delG mutation, as identified by ARMS-PCR (Antoniadi et al., 1999). One patient heterozygous for the GJB2 p.K224Q (c.670A>C) mutation and two patients heterozygous for the GJB2 p.R127H (c.380G>A) mutation were also excluded due to the still unclear association of these mutations with nonsyndromic hearing impairment.

Mutation screening of the GJB2 gene

Genomic DNA was extracted from blood lymphocytes by salting out procedure (Miller et al., 1988). All patients were initially screened for the presence of the c.35delG mutation by ARMS-PCR, as previously described (Antoniadi et al., 1999).

ARMS-PCR primers were designed for the p.L90P (c.269T>C) mutation for the direct screening of the families in which this mutation was originally found. The forward primer used was the CXL90P-F: 5′ - AAG GAG GTG TGG GGA GAT GAG CA -3′ and the modified reverse primer was the L90P-MUT: 5′ - TAG GCC ACG TGC ATG GCC ATT G -3′. An additional set of primers, primer 5: 5′ -AGT GCT GCA AGA AGA ACA ACT ACC - 3′ and primer 6: 5′ - CTC TGC ATC ATG GGC AGT GAG CTC - 3′ from the beta-globin gene (Old et al., 1990) was added for coamplification, to serve as an internal control of the PCR reaction. The amplification was performed in a total volume of 50 μL, containing 200 ng template DNA, 10 × buffer (200 mM Tris-HCl pH 8.4, 500 mM KCl), 1.5 mM MgCl₂, 0.2 mM each of deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate (dTTP), and 25 pmol of each primer. The initial denaturation was at 95°C for 3 min, followed by 30 cycles of 94°C for 1 min, 65°C for 1 min, 72°C for 1 min 30 s, and a final step of extension for 6 min at 72°C. The specific product for the mutation is 170 bp, whereas the control band is 323 bp.

Detection of the p.W24X (c.71G>A) mutation was performed by PCR amplification followed by restriction fragment length polymorphism (RFLP) analysis with the use of restriction endonuclease AluI. A fragment of 360 bp was amplified with the use of the following primers: forward: Cx48U 5′ - GGT GAG GTT GTG TAA GAG TTG G - 3′ (Abe et al., 2000) and reverse: 35delGcom 5′ - GAA GTA GTG ATC GTA GCA CAC GTT CTT GCA - 3′ (Scott et al., 1998). The PCR was performed in a total volume of 50 μL, containing 200 ng template DNA, 10× buffer (200 mM Tris-HCl pH 8.4, 500 mM KCl), 1.5 mM MgCl₂, 0.2 mM each of dATP, dCTP, dGTP, and dTTP, and 25 pmol of each primer, according to the following protocol: 94°C for 3 min, followed by 30 cycles of 94°C for 1 min, 62°C for 1 min, 72°C for 1 min, and a final step of extension for 5 min at 72°C; the product was subsequently digested with AluI. The enzyme recognizes an AGCT sequence in both the mutant and wild type allele and yields two fragments of 267 and 93 bp. The mutation causes a single nucleotide change (c.71G>A) that creates a new AluI restriction site (GGCT → AGCT) at nt 286-289 (GenBank, GI:195539329), and thus digestion of the mutant allele yields three fragments of 135, 132, and 93 bp.

We used a PCR-RFLP protocol to detect the p.delE120 (c.358-360delGAG) mutation using forward primer Con26nor: 5′-TGG GGC ACG CTG CAG ACG ATC CTG GGG AG-3′ and reverse primer delE120com: 5′-GAA GCC GTC GTA CAT GAC ATA GAA GAC GTA CAT-3′. The amplification was performed in a total volume of 50 μL, containing 200 ng template DNA, 10 × buffer (200 mM Tris-HCl pH 8.4, 500 mM KCl), 1.5 mM MgCl₂, 0.2 mM each of dATP, dCTP, dGTP, and dTTP, and 25 pmol of each primer. The initial denaturation was at 94°C for 3 min, followed by 30 cycles of 94°C for 1 min, 58°C for 1 min, 72°C for 1 min 30 s, and a final step of extension for 6 min at 72°C. The amplified fragment of 476 bp (nt 222-698, according to sequence GI:195539329, GenBank) was digested with restriction endonuclease Hpy188III. The mutation p.delE120 is a deletion of a GAG triplet at nt 358-360, which results in deletion of a Glu in the protein sequence. Digestion of the mutant allele yields two fragments of 348 and 128 bp.

For the detection of the p.R184P (c.551G>C) mutation, primers were designed to span a region of 203 bp (nt 633-836, according to sequence GI:195539329, GenBank). The PCR was performed in a total volume of 50 μL, containing 200 ng template DNA, 10 × buffer (200 mM Tris-HCl pH 8.4, 500 mM KCl), 1.5 mM MgCl₂, 0.2 mM each of dATP, dCTP, dGTP, and dTTP, and 25 pmol of each primer, according to the following protocol: 94°C for 3 min, followed by 30 cycles of 94°C for 1 min, 58°C for 1 min, 72°C for 1 min, and a final step of extension for 5 min at 72°C. The forward primer was R184P-F: 5′-ATC TTC TTC CGG GTC ATC TTC - 3′ and the reverse primer was R184P-R: 5′ - GAC ATT CAG CAG GAT GCA AAT - 3′. Restriction endonuclease HpaII digestion of the wild type allele yields three fragments of 123, 72, and 8 bp, whereas digestion of the mutant allele yields two fragments of 195 and 8 bp. The mutation causes a single nucleotide change (c.551G>C) that eliminates a HpaII recognition sequence (CCGG → CCCG) at nt 641-644 (GenBank, GI:195539329).

At this point, it should be indicated that although the tests for p.L90P, p.W24X, and p.delE120 are specific, the test for p.R184P (c.551G>C) is not. It could also detect another described variant, p.R184W (c.550C>T), given that both mutations eliminate the same HpaII restriction site. Hence, the test appears to be useful for detection but not for identification.

Results

The p.L90P and p.W24X mutations were not found in our panel of 28 familial cases. These mutations were previously identified in compound heterozygosity with the c.35delG mutation in three and two sporadic cases, respectively, and thus were characterized as recurrent in the Greek population (Pampanos et al., 2002). Mutation p.R184P was previously found in compound heterozygosity with c.35delG in one familial case and in one sporadic case, but no additional p.R184P chromosome was identified by our screening method in the group of 28 familial cases.

Results from all familial cases (n = 51) analyzed in this and previous studies of our group are presented in Table 1. A total of 18 patients were c.35delG homozygotes (35.3%). The mutation p.R127H was identified as the sole mutation in two cases (Antoniadi et al., 2000). The association of this mutation with nonsyndromic hearing impairment is still unclear (Estivill et al., 1998). Although it has been previously suggested that the p.R127H variant seems to be a frequent nonpathogenic variant in Gypsy populations (Minárik et al., 2003; Alvarez et al., 2005), our two cases with the p.R127H variant were not of Gypsy origin. The p.K224Q sequence variation was detected as the sole mutation in one case and has been previously reported by our group as a novel GJB2 mutation (Antoniadi et al., 2000). The p.delE120 mutation was found in compound heterozygosity with c.35delG in one case. In total, biallelic GJB2 mutations were detected in 20/51 (39.2%) of familial cases in our material. In one familial case, the c.35delG mutation was detected as the sole mutation in the proband with hearing loss and in his hearing father. There was history of deafness in the mother, her brother, and her father. No c.35delG mutation was found in the mother.

Table 1.

Mutations Identified in a Total of 51 Unrelated Greek Familial Cases of Sensorineural Prelingual Deafness by This and Previous Studies

Genotype	Cases
c.35delG/c.35delG	18
c.35delG/p.R184P	1
c.35delG/p.delE120	1
c.35delG/−	1
p.K224Q/−	1
p.R127H/−	2
−/−	27
Total	51

Results from control screening are shown in Table 2. The mutation p.L90P was present in 4/200 controls, suggesting that it might be frequent in the general Greek population. All controls were tested negative for mutations p.W24X, p.R184P, and p.delE120. Examples of the results obtained for the different detection tests are shown in Figure 1.

FIG. 1.

Examples of the results obtained for the different detection tests: lanes 1 and 11: PhiX174 DNA/HinfI marker; lanes 2 and 3: normal and heterozygote for the p.L90P mutation; lanes 4 and 5: normal and heterozygote for the p.W24X mutation; lanes 6 and 7: normal and heterozygote for the p.delE120 mutation; lanes 8 and 9: normal and heterozygote for the p.R184P mutation; lane 10: blank. The 8-bp band (lanes 8 and 9) is not visible in the agarose gel.

Table 2.

Prevalence of GJB2 Recurrent Mutations in the Healthy Greek Population

Mutation	Method	Prevalence
c.35delGa	ARMS-PCR	14/395 = 3.5%
p.L90P	ARMS-PCR	4/200 = 2.0%
p.W24X	PCR-RFLP	0/200
p.delE120	PCR-RFLP	0/200
p.R184P	PCR-RFLP	0/200

Antoniadi et al. (1999).

ARMS, amplification refractory mutation system; RFLP, restriction fragment length polymorphism.

Discussion

The majority of childhood deafness is nonsyndromic, and there is a great interest in identifying the underlying genes responsible as the primary approach for understanding this heterogeneous condition. After the identification of the GJB2 gene, which is responsible for a major proportion of nonsyndromic sensorineural prelingual deafness, a lot of sequence alterations have been reported in the effort to completely characterize the molecular basis of this frequent form of childhood deafness (The Connexin-deafness homepage, http://davinci.crg.es/deafness/).

In the current study, we present easy, rapid, and cost-effective screening techniques for other recurrent GJB2 mutations in the Greek population besides the frequent c.35delG mutation. In our material, 20/51 familial cases (39.2%) were identified with GJB2 disease causing mutations in both chromosomes, and the c.35delG mutation accounted for 95.0% of the GJB2 deafness mutations. We have previously reported that c.35delG homozygosity accounts for 27.3% of sporadic deafness cases in the Greek population (Pampanos et al., 2002). This is in concordance with other studies (Rabionet et al., 2000b) and also with our finding that there is a greater variation of GJB2 mutations in sporadic cases (we did not identify any p.L90P or p.W24X mutation in our panel of familial cases). Although we failed to identify the p.L90P mutation in our group of patients, we determined a frequency of 2% in healthy controls. This finding suggests that p.L90P appears to be the second most frequent GJB2 mutation in Greeks and could be offered in carrier screening. Our findings also indicate that the majority of familial cases of prelingual deafness are not due to biallelic GJB2 mutations, suggesting that other genes are responsible in such cases. Also, there is now increasing evidence that the manifestations of many genetic disorders are influenced by modifier genes distinct from the disease locus (Hilgert et al., 2009).

Greece is among the countries with a high carrier frequency of the c.35delG mutation, and other recurrent GJB2 mutations have now been identified in the Greek population, so this population could be screened at large. Taking into account that the number of public genetics laboratories in the country is limited and that direct sequencing of healthy persons tested negative for the c.35delG would be a procedure of high cost, we hereby suggest a two-step analysis for the healthy population: the initial c.35delG routine test followed by further analysis using protocols as described here for the subjects found negative for the c.35delG mutation.

Footnotes

Acknowledgment

This study was supported in part by a grant from Oticon Fonden, Denmark (to M.B.P.).

Disclosure Statement

No competing financial interests exist.

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